For growing axons to find their targets in the developing brain, their growth cones must respond to both attractive and repulsive signals in the environment. It has recently been shown that growth cones can modulate their responses to particular signal, even switching from attraction to repulsion; however, it is not known if this occurs in vivo. The zebrafish visual system is uniquely suited for studying how growth cones integrate positive and negative signals in vivo, and testing how their responses to signals change as they pathfind. The proposed experiments study the roles of two classes of guidance cues, Slits and netrins, in retinal axon pathfinding. Slits are thought to signal repulsively through the Astray/Robo2 receptor, while netrins are thought to signal attractively through the DCC receptor. Slit/Robo2 and netrin/DCC are both known to guide retinal axons, but their roles in different parts of the pathway are unknown. A combination of forward-genetic, reverse-genetic, and transgenic approaches will be used to perturb Slit/Robo2 and netrin/DCC signaling in vivo, to test where in the retinal pathway these signals are important, and to test whether their roles change over the course of the pathway. The zebrafish retinotectal system not only allows visualization of retinal axons in vivo with exquisite resolution, but also allows precisely targeted perturbations of their in vivo environment. The results and techniques developed here will help lead to a comprehensive understanding of all the signals that guide retinal axon growth, and how these different signals interact. In summary, this project will illuminate where, when, and how the guidance of zebrafish retinal axons requires Slit/Robo2 and netrin/DCC signaling. The resulting knowledge of genetic and developmental mechanisms will be important for understanding human diseases, such as albinism, that affect optic axon guidance. This knowledge will also be critical for designing therapies to reverse optic nerve degeneration. More generally, this project will test broad principles of axon guidance which are important for understanding the wiring of the nervous system and the basis of inherited neurological disease.